Multidimension cultivation analysis by standard and omics methods for optimization of therapeutics production

Abstract

Background: During the last decades Chinese Hamster Ovary (CHO) cells have been extensively used for research and biotechnological applications. About 40% of newly approved glycosylated protein pharmaceuticals are produced in CHO cells today [1]. Despite the increasing relevance of these cells for biopharmaceutical production little is known about effects of intracellular processes on productivity and product quality. In the last years supplementation of serum-free media with insulin - more and more replaced by IGF-1 and its analogue LongR3 - was utilized to enhance product titer and quality. To compare the intracellular effects of these two supplements an antibody producing CHO cell line was cultivated in batch mode using insulin, LongR3 or no growth factor as reference. Subsequently, different omics-techniques were applied to analyze medium and cell samples. Material(s) and Method(s): CHO cells producing an antibody were cultured in chemically defined serum-free medium TC-BN.CHO (Teutocell AG) with addition of 6 mM glutamine. Three cultivations (37degreeC, pH 7.1, 40% DO, 120 rpm) were performed in 2l-bioreactor systems with supplementation of 10 mg/l insulin or 0.1 mg/l LongR3. The third culture was untreated and served as reference. Samples were taken every 24 h. Viable cell density and cell viability were measured using Cedex (Roche). Glucose and lactate were determined via YSI 2300 STAT PlusTM Glucose & Lactate Analyzer (YSI Life Science). Quantitation of antibody production was determined using POROS A columns (Invitrogen). N-Glycan abundance was analyzed by HPAEC-PAD method [2]. For RNA samples 'Total RNA NucleoSpin Kit' (Macherey-Nagel) was used. Quality and quantity of RNA were determined using Nano Drop 1000 (Peqlab) and Bioanalyzer (Agilent). An in-house developed customized cDNA microarray with 41,304 probes was applied for transcriptome analysis. RNA was labeled using Agilent LIQUA Kit, one-color. Processing of microarray data was performed in ArrayLims and EMMA2 [3]. Raw data were standardized using Feature Extractor (Agilent) and LOWESS normalization. Result(s): Cultivation data illustrated that maximal cell density was higher in cultivations with insulin and LongR3 compared to that without growth factor. Additionally, glucose consumption and lactate production was slightly higher in cultivations with these supplements but time point of glutamine depletion was similar in all reactors after similar cultivation time (Figure 1A). Furthermore, product quantity and product quality was not influenced by growth factor addition. The most abundant glycoforms after 7 days of cultivation were G0F with about 50% and G1F with about 40% in all cultivation set-ups (Table 1). For transcriptome analysis samples on day 5 were compared with those on day 3. Therefore, the following settings were used in statistical tests: a twosample t-test with a p-value = 6 (for A1 or A2) and intensity ratio >= 0.6 or